Structural dynamics of the cooperative binding of organic molecules in the human cytochrome P450 3A4
Structural dynamics of the cooperative binding of organic molecules in the human cytochrome P450 3A4
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DOI：
10.1021/ja066007j
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发表时间：
2007-02-14
影响因子：
15
通讯作者：
Nussinov, Ruth
中科院分区：
文献类型：
--
作者：
Fishelovitch, Dan;Hazan, Carina;Nussinov, Ruth
细胞色素 P450 3A4 (CYP3A4) 是一种关键酶，负责 50% 的口服药物的代谢，其表现出有趣的动力学行为，其典型特征是反应速度对底物浓度的 S 形依赖性。有证据表明两种底物在酶的活性位点结合，但这种协同结合的机制尚不清楚。地西泮是一种通过 CYP3A4 进行 S 形依赖性代谢的药物。代谢是通过从药物中提取氢原子来启动的。为了了解决定抽提过程中 C-H 键的协同结合和并置的因素，我们对两种酶构象异构体进行了分子动力学模拟，并用一个和两个地西泮分子检查了无底物酶和结合酶之间的差异。我们的结果表明，效应底物与活性底物和酶都相互作用，并且这种相互作用导致侧链重新定向，而远程效应相对较小。根据实验，我们发现F304位于活性结合位点和效应结合位点之间的界面，是协同结合机制中的关键残基。效应底物的添加稳定了 F304 及其环境，尤其是 F213，并诱导活性底物的有利方向，导致提取的目标氢与酶的活性物质之间的距离较短。此外，在酶的一种构象异构体中，残基 R212 可能与 F304 强烈相互作用并抵消效应物对酶的影响。
Cytochrome P450 3A4 (CYP3A4) is a key enzyme responsible for the metabolism of 50% of all orally administered drugs which exhibit an intriguing kinetic behavior typified by a sigmoidal dependence of the reaction velocity on the substrate concentration. There is evidence for the binding of two substrates in the active site of the enzyme, but the mechanism of this cooperative binding is unclear. Diazepam is such a drug that undergoes metabolism by CYP3A4 with sigmoidal dependence. Metabolism is initiated by hydrogen atom abstraction from the drug. To understand the factors that determine the cooperative binding and the juxtaposition of the C-H bond undergoing abstraction, we carried out molecular dynamics simulations for two enzymatic conformers and examined the differences between the substrate-free and the bound enzymes, with one and two diazepam molecules. Our results indicate that the effector substrate interacts both with the active substrate and with the enzyme, and that this interaction results in side chain reorientation with relatively minor long-range effects. In accord with experiment, we find that F304, in the interface between the active and effector binding sites, is a key residue in the mechanism of cooperative binding. The addition of the effector substrate stabilizes F304 and its environment, especially F213, and induces a favorable orientation of the active substrate, leading to a short distance between the targeted hydrogen for abstraction and the active species of the enzyme. In addition, in one conformer of the enzyme, residue R212 may strongly interact with F304 and counteract the effector's impact on the enzyme.